NEKODIRAJOČE RNA KOT MOŽNI GENETSKI KAZALCI PREŽIVETJA PRI BOLNIKIH Z GLIOMI

Matos, Boštjan (author), Glavač, Damjan (mentor)

Uvod Rak je kompleksna genetska bolezen, ki je posledica kopičenja škodljivih mutacij. Te lahko vplivajo na spremenjeno izražanje genov, kar vodi v napačno količino ali obliko njihovih proteinskih produktov, ki lahko povzročijo spremembo biokemijskih poti. Slednje omogočajo karcinogenezo oziroma rast in preživetje rakavih celic na podlagi neovirane delitve, neodzivnosti na signale celičnega okolja in izgube sposobnosti apoptoze. V zadnjem obdobju je veliko raziskav usmerjenih v preučevanje nekodirajočih področij genoma, katerih glavna funkcija je uravnavanje izražanja genov. Najpomembnejšo skupino predstavljajo nekodirajoče RNA (ncRNA), kamor uvrščamo tudi dolge nekodirajoče RNA (lncRNA) in mikro RNA (miRNA). MiRNA so približno 22 nukleotidov dolge molekule, ki z vezavo na mRNA preprečijo tvorbo proteinov ali povzročajo razgradnjo mRNA; lncRNA so daljše, in sicer od 200 do nekaj tisoč nukleotidov. Njihovo izražanje in delovanje vpliva na proces diferenciacije gliomov. Hipoteza Predpostavljamo, da določene lncRNA kažejo spremenjen vzorec izražanja tudi pri bolnikih z gliomi in da se ti vzorci razlikujejo med gliomi različnih stopenj malignosti. Glede na razlike v izražanju lncRNA predpostavljamo, da lahko dopolnimo klasifikacijo različnih histoloških podtipov gliomov. Predpostavljamo, da bo spremenjeno izražanje miRNA glede na prejeto obliko onkoterapije različno glede na vrsto onkološkega zdravljenja in da bo to vplivalo na preživetje bolnikov z GBM. Metode Za prvi del naloge smo analizirali izražanje 90 lncRNA pri 64 bolnikih z malignimi gliomi astrocitne in oligodendroglialne diferenciacije. V ta namen smo uporabili sveže vzorce tumorskega tkiva, shranjenega v RNAlater takoj po biopsiji. Za drugi del naloge smo preučevali izražanje 11 miRNA (miR-9, miR-15b, miR-21, miR-26b, let-7a, let-7b, let-7d, let-7f, miR-7, miR-124 in miR-199a) glede na različno obliko prejete onkološke terapije (RT ali RT in KT) pri bolnikih z rekurentnim GBM. Vključili smo 83 bolnikov, ki so bili zaradi ponovitve tumorja operirani dvakrat. Vzorci, pridobljeni pri prvi (prva biopsija) in drugi operaciji (druga biopsija), so bili za namene patohistoloških preiskav fiksirani v formalinu in vklopljeni v parafin. Za analizo izražanja miRNA in lncRNA smo uporabili metodo qPCR v realnem času. Rezultati V prvem delu raziskave smo dokazali signifikantno spremenjeno izražanje več lncRNA pri vseh podtipih malignih gliomov. Pri vseh smo ugotovili znižano izražanje MEG3, JPX, RNCR3 in zvišano izražanje HOTAIR in ZFAS1. V drugem delu raziskave smo pri parnih vzorcih GBM ugotovili statistično pomembno spremembo v izražanju sedmih miRNA (miR-7, miR-9, miR-21, miR-26b, miR-124a, miR-199a in let-7f). Izražanje miR-15b, let-7d in let-7f se je statistično pomembno razlikovalo v vzorcih z rekurentnim tumorjem, ko smo primerjali tiste, ki so bili zdravljeni z RT in KT, s tistimi, ki so prejeli samo RT. Čas do ponovitve tumorske razrasti je bil povprečno sedem mesecev pri bolnikih, ki so prejemali le RT po prvi operaciji, in v povprečju enajst mesecev pri bolnikih, ki so prejemali RT in KT. Po drugi operaciji je bil čas preživetja statistično značilno daljši (osem mesecev) pri bolnikih, ki so prejemali KT po drugi operaciji, v primerjavi s tistimi, ki niso bili zdravljeni (pet mesecev). Zaključki Ugotovili smo, da se izražanje izbranih lncRNA pomembno razlikuje med posameznimi podtipi gliomov različnih stopenj malignosti. Ugotovili smo, da se izražanje izbranih miRNA razlikuje med vzorci GBM po prvi in drugi operaciji ter da se izražanje nekaterih izbranih miRNA spreminja glede na vrsto prejete onkološke terapije. Rezultati naše raziskave kažejo, da je čas do ponovitve tumorske rasti daljši pri bolnikih, ki so bili po operaciji zdravljeni z RT in s KT, v primerjavi s tistimi, ki so bili zdravljeni samo z RT. Izbrana skupina bolnikov je bila omejena na bolnike z GBM, ki so bili po kliničnih standardih (status Karnofsky več kot 80) sposobni reoperacije, zato lahko rezultate izražanja miRNA in klinični potek (čas do ponovitve tumorja, preživetje) pripišemo izključno tej skupini bolnikov, in ne na splošno bolnikom z GBM. Menimo, da bi lahko izražanje določenih lncRNA in (ali) miRNA uporabili kot dodatno informacijo pri objektivnejši postavitvi diagnoze ter morda tudi pri napovedi bolezni in razvoju novih načinov zdravljenja. Glede tega menimo, da bi nekatere miRNA lahko bile genetski označevalci pri napovedi tumorske ponovitve in da so potencialno vpletene v napredovanje tumorja.

• Language: Slovenian
• Keywords: gliomi, radioterapija, kemoterapija, nekodirajoča RNA, dolga nekodirajoča RNA, mikro RNA, izražanje, preživetje
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2019
• PID: 20.500.12556/RUL-106007
• COBISS.SI-ID: 298524160
• Publication date in RUL: 13.01.2019

Secondary language

• Language: English
• Title: NON-CODING RNAS AS POSSIBLE GENETIC BIOMARKERS OF SURVIVAL IN GLIOMA PATIENTS

Abstract

Background Cancer is a complex genetic disease caused by the accumulation of harmful mutations. They may affect the altered gene expression, leading to incorrect amount or form of their protein products. Thus, they modified biochemical routes or allow carcinogenesis, growth and survival of cancer cells through the unobstructed division of cancer cells, non-response to signals from the cellular environment and the loss of the ability of apoptosis. Recently, many studies have focused on non-coding regions of the genome research, whose main function is to regulate gene expression. The most important group consists of non-codingRNA (ncRNA), which include long non-codingRNA (lncRNA) and microRNA (miRNA). MiRNA is approximately 22 nucleotides long ncRNA that binds to the mRNA preventing the formation of protein or degradation of mRNA. LncRNA are long from 200 up to several thousand nucleotides. Their expression and performance impact the process of differentiation of glioma. Hypothesis We assume that an analysis of the expression of ncRNA can more accurately classify the different histological types of gliomas. Expression of lncRNAs can provide additional information for distinguishing different stages of malignancy. The expression of certain miRNAs in recurrent GBM changes after oncological therapy and depends on the type of oncological treatment used. The survival after second surgery depends on the type of oncological therapy received. Methods In the first part of our study we analyzed the expression of several lncRNAs in 64 patients diagnosed with malignant glioma of astrocytic or oligodendroglial differentiation. For second part of the study we used paired paraffin embedded tissues from 83 patients with primary GBM, obtained from newly diagnosed and corresponding recurrent tumour. Expression of 11 miRNAs (miR-7, miR-9, miR-15b, miR-21, miR-26b, miR-124, miR-199a, let-7a, let-7b, let-7d, let-7f) was analysed before and after oncological treatment. Quantitative PCR method was used. Results Our results showed downregulation of MEG3, JPX and RNCR3 and upregulation of HOTAIR and ZFAS1 in all glioma subtypes. The analysis showed a statistically significant change in the expression of 7 miRNAs (miR-7, miR-9, miR-21, miR-26b, miR-124a, miR-199a and let-7f) in biopsy specimen of recurrent GBM compared with GBM at first biopsy. Expression of miR-15b, let-7d and let-7f was statistically significantly different in recurrent GBM samples when we compared those who were treated with radiotherapy and chemotherapy with those who received radiotherapy only. Time to recurrence of tumor growth was on average 7 months in patients who received radiotherapy after the first surgery, and on average of 11 months for patients who received radiation therapy and chemotherapy. After the second surgery the survival time was significantly longer (8 months) in patients who received chemotherapy compared with those that have not been treated (5 months). Conclusions We have found that the expression of selected lncRNA differs significantly between the individual subtypes of the gliomas of the various stages of malignancy. We have found that expression of selected miRNA varies between GBM samples after first and second surgery. We also found that the expression of some selected miRNA varies according to the type of oncology therapy. The results of our study show that the time to recurrence of tumor growth is longer in patients who were treated with radiotherapy and chemotherapy after surgery compared to patients treated with radiotherapy alone. The selected patient group was limited to GBM patients who were capable of reoperating according to clinical standards (Karnofsky status over 80), therefore the results of the expression of miRNA and the clinical course (time to tumor recurrence, survival) can be attributed exclusively to this group of patients and not generally to GBM patients. We believe that the expression of certain lncRNA and/or miRNA could be used as additional information in a more objective diagnosis, and possibly also in predicting disease and developing new therapies. In this regard, we believe that some miRNA could serve as potentially genetic markers in predicting tumor recurrence and are likely involved in tumor progression.

• Keywords: gliomas, radiotherapy, chemotherapy, noncoding RNA, long-noncoding RNA, micro RNA, expression, survival